WO2011028890A2 - Compositions et procédés de traitement de la douleur névropathique - Google Patents

Compositions et procédés de traitement de la douleur névropathique Download PDF

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WO2011028890A2
WO2011028890A2 PCT/US2010/047657 US2010047657W WO2011028890A2 WO 2011028890 A2 WO2011028890 A2 WO 2011028890A2 US 2010047657 W US2010047657 W US 2010047657W WO 2011028890 A2 WO2011028890 A2 WO 2011028890A2
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sip30
seq
nucleic acid
substance
group
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WO2011028890A3 (fr
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Lei Yu
Ning Guo
Yu-Qui Zhang
Zhi-Qi Zhao
Naihe Jing
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Rutgers, The State University Of New Jersey
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Priority to CN201080049104.1A priority Critical patent/CN102666877B/zh
Priority to EP10814485A priority patent/EP2473635A4/fr
Priority to US13/393,399 priority patent/US9068984B2/en
Priority to CA2772931A priority patent/CA2772931A1/fr
Publication of WO2011028890A2 publication Critical patent/WO2011028890A2/fr
Publication of WO2011028890A3 publication Critical patent/WO2011028890A3/fr
Priority to IL218448A priority patent/IL218448A0/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/48—Nerve growth factor [NGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2842—Pain, e.g. neuropathic pain, psychogenic pain

Definitions

  • the present invention relates to the treatment of neuropathic pain in a mammalian using SIP30 antagonists.
  • Neuropathic pain is a complex, chronic pain state that is generally accompanied by tissue injury. With neuropathic pain, the nerve fibers themselves might be damaged, dysfunctional, or injured. These damaged nerve fibers send incorrect signals to other pain centers.
  • the clinical causes of neuropathic pain are widespread and include both trauma and disease. For example, traumatic nerve compression or crush and traumatic injury to the brain or spinal cord are common causes of neuropathic pain. Furthermore, most traumatic nerve injuries also cause the formation of neuromas, in which pain occurs as a result of aberrant nerve regeneration.
  • cancer-related neuropathic pain is caused when tumor growth painfully compresses adjacent nerves, brain or spinal cord. Neuropathic pain is associated with diseases such as diabetes or alcoholism.
  • neuropathic pain often do not provide the patients in need of such treatment the adequate pain relief.
  • current therapies have serious side-effects including, for example, cognitive changes, sedation, nausea and, in the case of narcotic drugs, addiction.
  • Many patients suffering from neuropathic pain are elderly or have other medical conditions that particularly limit their tolerance of the side-effects associated with available drug therapy.
  • a number of anti-inflammatory, anxiolytic, narcotic and even anti-convulsants are currently used by the practitioners to treat neuropathic pain, but with limited success.
  • the inadequacy of current therapy in relieving neuropathic pain calls for new compositions and methodologies of addressing the physical and social needs of the patient suffering from such condition. Methods of alleviating neuropathic pain would improve the quality of life for many people suffering from pain due to trauma or disease .
  • the present invention fills the foregoing need by providing compounds, compositions, methods and systems for treating neuropathic pain regulated by SIP30. Applicants have surprisingly found that SIP30 antagonists are effective in treating neuropathic pain.
  • the present invention also provides a screening method comprising: providing a sample containing at least a portion of an SIP30 polypeptide; adding a test compound to at least a first portion of the sample; comparing at least one parameter from at least the first portion of the sample with the at least one parameter from at least a second portion of the sample, wherein at least the second portion of the sample does not include the test compound.
  • Applicants invention also process of determining a substance for its ability to interact with SIP30-like molecule protein comprising: a) providing a SIP30-like molecule polypeptide comprising the contiguous amino acid sequence of any from the group of SEQ ID Bl through SEQ ID B130; and b) testing the ability of said substance to interact with a SIP30-like molecule .
  • Figure 1 illustrates rapid onset of thermal hyperalgesia and mechanical allodynia in rats receiving CCI surgery. Time courses for the development of mechanical allodynia (A) and thermal hyperalgesia (B) following chronic constriction injury of sciatic nerve were shown. p ⁇ 0.05 vs. non-CCI injury paw.
  • Figure 2 illustrates amino acid sequence alignment of mammalian SIP30 orthologues shows high degree of sequence homology, implicating similar function. Amino acid sequences of all mammalian SIP30 available in public domain are aligned. Gaps in alignment are shown as blank space. Amino acid residue identity with that of the corresponding human SIP30 sequence is indicated by a dash (-) . Underline: putative coiled-coil domain.
  • the 2 kb mRNA band of SIP30 was marked with an arrow, and 18S ribosomal RNA was used as an internal control for sample loading. Source of tissue for RNA was indicated across the top of the gel for each lane.
  • C SIP30 expression in the spinal cord.
  • FIG. 4 illustrates SIP30 was up-regulated during CCI .
  • A SIP30 mRNA levels in spinal cord (upper panel) and DRG (lower panel) .
  • Real-time PCR amplification results for SIP30 mRNA are shown relative to the mean levels in normal animals.
  • Spinal cord tissues from rats receiving no surgery (normal), sham-CCI surgery (sham), or CCI surgery (3 days and 7 days post surgery, respectively) were collected, and RNA was isolated from the ipsilateral and contralateral side of the sham or CCI surgery for real-time PCR analysis.
  • Significant difference p ⁇ 0.05 vs. normal or sham group was observed in the ipsilateral but not contralateral spinal cord for both day 3 and day 7 after CCI surgery.
  • SIP30 localization in CCI spinal cord (a) SIP30 (red) is co-localized with NeuN (green) , a neuronal marker, in the spinal dorsal horn, 3 days after CCI (20x) . (b) Enlarged image of boxed area in (a) as superimposed image (left panel) or separate images (right top and bottom panels) (40x) . (c) Double immunofluorescence staining shows the SIP30 (red) is not co- localized with GFAP (green) in the dorsal horn 3 days after CCI .
  • Double immunofluorescence staining shows the SIP30 (red) is not co-localized with OX-42 (green) in the dorsal horn 3 days after CCI. SIP30 did not show overlap staining with either GFAP or OX-42.
  • FIG. 5 illustrates SIP30 participates in the regulation of development and maintenance of peripheral nerve in ury-induced neuropathic pain.
  • AS ODN antisense oligonucleotide
  • FIG. 5 illustrates SIP30 participates in the regulation of development and maintenance of peripheral nerve in ury-induced neuropathic pain.
  • A Following the intrathecal injection of SIP30 antisense oligonucleotide (AS ODN) for 4 days (from day 0 to day 3 post-CCI), the development of mechanical allodynia (a) and thermal hyperalgesia (b) was reduced in ipsilateral paw to CCI ("Ipsi.”), whereas NS and MM ODN had no effect. p ⁇ 0.05 vs. either NS or MM group.
  • c, d No change was seen in contralateral paw ("Cont.") after the same treatment.
  • FIG. 6 illustrates anti-SIP30 antisense oligonucleotide knock down of SIP30 and SNAP25 levels.
  • SIP30 mRNA was increased after CCI, and the increase was reduced by antisense oligonucleotide (ODN) .
  • ODN antisense oligonucleotide
  • AS SIP30 antisense
  • MM missense
  • Rat were sacrificed and the lumbar spinal cords were dissected at six hours after the last injection.
  • Real-time PCR amplification for SIP30 mRNA showed a significant decrease in ipsilateral spinal cord in CCI rats receiving SIP30 AS ODN compared with animals receiving MM ODN (p ⁇ 0.05).
  • the dotted line indicates mRNA level in sham CCI animals.
  • Top panel representative Western blots of SIP30 protein. ⁇ -Actin was used as the internal control.
  • Bottom panel quantitative results of SIP30 protein.
  • the dotted line indicates protein level in sham ones. significant difference from NS or MM (p ⁇ 0.05) .
  • NS normal saline
  • AS ODN antisense oligodeoxynucleotides
  • MM missense
  • day (0-3) rats were injected intrathecally with AS, MM, or NS from day 0 (6 hrs before CCI operation) to day 3 post-CCI; day (4-7), rats were injected intrathecally with AS, MM, or NS from day 4 to day 7 post-CCI.
  • C Real-time PCR amplification for SNAP25 mRNA showed a significant increase in ipsilateral spinal cord after CCI operation. p ⁇ 0.05 vs. sham group.
  • Intrathecal injection of SIP30 AS ODN every 24 hours for 4 times caused a trend to decrease in SNAP25 mRNA in ipsilateral spinal cord of CCI rats, although that did not reach statistical significance.
  • (D) Real-time PCR amplification for PSD95 mRNA showed a significant increase in ipsilateral spinal cord after CCI operation. p ⁇ 0.05 vs. sham group. Intrathecal injection of SIP30 AS ODN every 24 hours for 4 times did not affect PSD95 mRNA levels.
  • allodynia refers to a painful response to a usually non-painful stimulus and can be either static or mechanical. The pathophysiology of allodynia is thought to differs from referred pain, but can occur in areas other than the one stimulated.
  • gene refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or its precursor.
  • the polypeptide can be encoded by a full length coding sequence (either genomic DNA or cDNA) or by any portion of the coding sequence so long as the desired activity is retained.
  • gene also refers to an mRNA sequence or a portion thereof that directly codes for a polypeptide or its precursor.
  • transfection refers to the uptake of foreign DNA by a cell.
  • a cell has been "transfected” when exogenous (i.e., foreign) DNA has been introduced inside the cell membrane.
  • Transfection can be either transient (i.e., the introduced DNA remains extrachromosomal and is diluted out during cell division) or stable (i.e., the introduced DNA integrates into the cell genome or is maintained as a stable episomal element) .
  • Co-transfection refers to the simultaneous or sequential transfection of two or more vectors into a given cell.
  • promoter element refers to a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence.
  • a promoter sequence is, in general, bounded at its 3 ' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. Within the promoter sequence may be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • the promoter may be operably associated with other expression control sequences, including enhancer and repressor sequences.
  • operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced .
  • vector refers to a nucleic acid assembly capable of transferring gene sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • expression vector refers to a nucleic acid assembly containing a promoter which is capable of directing the expression of a sequence or gene of interest in a cell.
  • Vectors typically contain nucleic acid sequences encoding selectable markers for selection of cells that have been transfected by the vector.
  • vector construct refers to any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • antibody refers to a whole antibody, both polyclonal and monoclonal, or a fragment thereof, for example a F(ab)2r Fab, FV, VH or VK fragment, a single chain antibody, a multimeric monospecific antibody or fragment thereof, or a bi- or multi-specific antibody or fragment thereof.
  • the term also includes humanized and chimeric antibodies .
  • SIP30 refers to a nucleic acid sequence that encodes a 266 amino acid protein SNAP25 interacting protein of 30 kDa. SIP30 is expressed abundantly in nerve tissues and slightly in testis and kidney.
  • SIP30- related refers to any of a nucleic acid sequence listed in SEQ ID Al through SEQ ID A130.
  • SIP30 antagonist refers to any substance, compound and compositions, synthetic or natural, including but not limited to antisense nucleic acid sequences, siRNA, antibodies, small molecular entities, alone or in combination that can prevent, inhibit, reduce or neutralize the expression and activity of SIP30, directly or indirectly modulating the activity of neuropathic pain inducing intermediaries "NPII.”
  • SNAREs refers to one type of NPII, which is a soluble N-ethylmaleimide-sensitive factor attachment protein receptors, including but not limited to synaptosome-associated proteins of 25 kDa (SNAP25), syntaxins, and vesicle-associated membrane proteins (VAMP) , which are essential for regulated exocytosis of synaptic vesicles in neurotransmission.
  • treating or “treatment” of a disease refers to executing a protocol, which may include administering one or more drugs to a patient (human or otherwise) , in an effort to alleviate signs or symptoms of the disease. Alleviation can occur prior to signs or symptoms of the disease appearing, as well as after their appearance. Thus, “treating” or “treatment” includes “preventing” or “prevention” of disease. In addition, “treating” or
  • treatment does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols which have only a marginal effect on the patient .
  • patient refers to a biological system to which a treatment can be administered.
  • a biological system can include, for example, an individual cell, a set of cells (e.g., a cell culture), an organ, a tissue, or a multi ⁇ cellular organism.
  • a patient can refer to a human patient or a non-human patient.
  • hyperalgeisa refers to an increased sensitivity to pain, which may be caused by damage to nociceptors or peripheral nerves.
  • the present invention is directed to compounds, compositions, methods and systems for treating neuropathic pain regulated by SIP30.
  • SIP30 antagonists are effective in treating neuropathic pain.
  • SPI30 antagonists including but not limited to nucleotides, small interfering RNA (siRNA) molecules, natural or synthetic compounds that correspond to at least a portion of SPI30 nucleic acid sequence are effective in inhibiting the expression of SIP30, directly, or indirectly through modulating the activity of NPII and thereby providing a means for treating pain caused thereby.
  • small molecules capable of altering the configuration of SIP30 dependent intermediary proteins, thereby antagonizing the expression of SIP30 may be accomplished by siRNAs that have a specific substrate binding site which is complementary to one or more of the target nucleic acid regions or (i.e., able to base-pair with) a portion of a target RNA.
  • a specific substrate binding site which is complementary to one or more of the target nucleic acid regions or (i.e., able to base-pair with) a portion of a target RNA.
  • complementarity is 100%, but can be less if desired, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • one aspect of the present invention provides the use of DNA fragments, peptides, compounds, compositions thereof in the effected area to antagonize the expression and/or activity of SIP30.
  • Another aspect of the present invention is directed to synthetic or natural compounds that antagonize the activity of NPII.
  • Yet another aspect of the invention is directed to implant depots to deliver a suitable molecule to the area of interest triggering neuropathic pain.
  • At least one aspect of the invention is directed to the nucleic acid molecules corresponding to at least a portion of a neuropathic pain inducing nucleic acid sequences capable of inhibiting expression of NPII or any related pain inducing cytokines in a cell.
  • the inventors propose methods of treatment of pain for neuropathic pain conditions, including spinal cord-mediated pain, peripheral nerve damage related pain, herniated disc related pain, Carpel tunnel syndrome, multiple sclerosis, fibromyalgia, herpes zoster, HIV-related neuropathies, painful traumatic mononeuropathy, painful polyneuropathy (particularly due to diabetes), central pain syndromes (potentially caused by virtually any lesion at any level of the nervous system) , post-surgical pain syndromes (eg, post- mastectomy syndrome, post-thoracotomy syndrome, phantom pain) , and complex regional pain syndrome (reflex sympathetic dystrophy and causalgia.
  • spinal cord-mediated pain including spinal cord-mediated pain, peripheral nerve damage related pain, herniated disc related pain, Carpel tunnel syndrome, multiple sclerosis, fibromyalgia, herpes zoster, HIV-related neuropathies, painful traumatic mononeuropathy, painful polyneuropathy (particularly due to diabetes), central pain syndromes (potentially caused
  • Another aspect of the present invention is directed to an expression vector comprising at least one DNA sequence encoding a siRNA molecule corresponding to at least a portion of a SIP30 nucleic acid sequence capable of inhibiting expression pain intermediaries in a cell operably linked to a genetic control element capable of directing expression of said siRNA molecule in a host cell.
  • Another aspect of the present invention is directed to a method for treating a subject suffering from neuropathic pain comprising administering into said subject at least a compound selected from the group consisting of an antisense sequence, a siRNA molecule, a peptide, a small molecule that corresponds and antagonizes the activity of at least a portion of a SIP30 nucleic acid sequence and or a SIP30 protein .
  • Another aspect of the present invention is directed to a system for treating a patient suffering from neuropathic pain comprising at least one antisense or siRNA molecule, a peptide, a small molecule alone or in combination with each other or further in combination with other neuropathic pain therapies and modalities and finally a means for introducing said compounds to the desired tissue of the patient.
  • candidate compounds including antisense, siRNA molecule that corresponds to at least a portion of a the SIP30 nucleic acid sequence can be introduced into the desired tissue by means of an injection, a pump or a depot .
  • a depot implant is implanted in a subject at or near a target site.
  • sites include an inflamed nerve, constricted nerve at a cerebral or a spinal site, or the surrounding soft tissue.
  • the siRNA depot implant is positioned in the affected areas. Additional embodiments of the invention provide for positioning the drug depot implant in the shoulder, hip, other joints or spine of a patient.
  • a targeted delivery system of one or more SIP30 antagonist molecules conveniently employ a catheter.
  • the targeted delivery system employs a syringe.
  • the targeted delivery system comprises a drug depot implant system administered locally by insertion of a catheter at or near a target site, the catheter having a proximal end and a distal end, the distal end having an opening to deliver a SIP30 antagonist in situ, the proximal end being fluidly connected to a pharmaceutical delivery pump.
  • the proximal end of the catheter may deliver the molecule to within 10 cm of a target site, more particularly, to within 5 cm of the target site.
  • SIP30 antagonist may inhibit other pain intermediaries mediated by TNF- , IL-1, IL-6 and other pain inducing cytokines .
  • the suitable compounds further comprises a modified release pharmaceutical composition.
  • the system can further comprise two or more combination of molecules.
  • a catheter is provided rather than a depot.
  • a catheter has a proximal end and a distal end, the distal end having an opening to deliver a pharmaceutical in situ, the proximal end being fluidly connected to a pharmaceutical pump .
  • the present invention is more specifically drawn to compositions and methods for the treatment of pain arising from inventors' discovery that inhibition of SIP30 results in reduction of pain of a neuropathic pain nature.
  • a SIP30 antagonist or a related molecule can be administered directly to a nerve or a tissue position near a nerve centrally or locally that is responsible of origination of neuropathic pain.
  • neuropathic pain conditions including spinal cord- mediated pain, compression injuries, peripheral nerve damage related pain, herniated disc related pain, Carpel tunnel syndrome, multiple sclerosis, fibromyalgia, herpes zoster, HIV-related neuropathies, painful traumatic mononeuropathy, painful polyneuropathy (particularly due to diabetes), central pain syndromes (potentially caused by virtually any lesion at any level of the nervous system) , post-surgical pain syndromes (eg, post-mastectomy syndrome, post- thoracotomy syndrome, phantom pain) , and complex regional pain syndrome (reflex sympathetic dystrophy and causalgia.
  • a substance to antagonize SIP30 or a related molecule can be administered directly to a nerve or a tissue position near a nerve.
  • neuropathic pain relief can be achieved by antagonizing a molecule related to SIP30; such a molecule can be a synaptic vesicle-related protein, and/or a molecule generally known as a SNAREs (Ungar and Hughson, 2003; Brunger, 2005) .
  • the present invention provides an advantageous strategy for reducing any type of pain associated with any medical condition or caused by any procedure that could induce or express any portion of the SIP30.
  • SIP30 is abundantly available in certain mammalian tissues. In brain, SIP30 is highly expressed in the inferior and superior colliculi, which contain important relay nuclei of the auditory and visual systems.
  • GST-pull-down and immunoprecipitation assays showed direct binding of SIP30 to SNAP25. Although SIP30 does not directly interact with syntaxin based on pull-down assays, syntaxin does co- immunoprecipitate with SIP30 suggesting that syntaxin is indirectly associated with SIP30, perhaps through SNAP25.
  • the inventors of the present invention disclose compounds and methods for reducing neuropathic pain associated with peripheral nerve injury.
  • localized delivery of an SIP30 antagonist will prevent, reduce or at least minimize the majority of negative side effects of systemic agents.
  • local delivery of SIP30 antagonist molecules can be used to achieve high concentrations of the SIP30 antagonist at the intended target site while using a low dose and minimizing risk of systemic side effects.
  • another aspect of the present invention embrace protracted release of the agent for long term relief of neuropathic pain by incorporating SIP30 antagonists alone or in combination with suitable anticonvulsant, anti- inflammatory, compounds targeting different ion channels, opioids, Na channel blockers, antidepressents , or anesthetic, more specifically, including but not limited to Mexiletine, Lidocaine, Tramadol, Morphine, Alfentanil, Ketamine, Methylprednisone, Adenosine, Glycine antagonists, Desipramine, Venlafaxine, gabapentin, into a biodegradable depot implant that will act as a depot for localized or central release of the agent.
  • Another aspect of the present invention provide reagents, methods and systems for inhibiting expression of NPII or cytokines responsible for inducing neuropathic pain in a cell using antisense, or siRNA molecules that correspond to at least a portion of SIP30 cytokine nucleic acid sequence of SNAREs.
  • SIP30 antagonists targeted to NPII and related cytokine mRNA are effective in inhibiting expression of neuropathic pain inducing cytokine, thereby providing improved methods for treating pain in a subject.
  • the methods of this aspect of the present invention can be performed utilizing routine techniques in the field of molecular biology. Basic texts disclosing general molecular biology methods include Sambrook et al .
  • the invention relates to isolated nucleic acid molecules comprising a nucleic acid sequence encoding complementary to nucleotides 11-31 of the coding region of the SNAP25 inteacting protein (GenBank accession number BC063144) .
  • the isolated nucleic acid molecule encodes proteins that neutralizes the up- regulation of SIP30 associated with neuropathic pain phenomenon.
  • the invention is directed to vectors comprising these nucleic acid molecules, as well as host cells comprising the vectors.
  • the invention relates to the proteins themselves.
  • the invention relates to antibody that is specific for SIP30 proteins and SNAREs including rat SNAP and all homologous or orthologous sequences for mammalian SIP30 as shown in Figure 2.
  • the antibody is a polyclonal antibody.
  • the antibody is a monoclonal antibody .
  • the invention relates to method of reducing the upregulation of SIP30 locally or centrally in specific neuropathic pain originating tissues, for example ipsilateral side of the spinal cord.
  • the isolated nucleic acid molecule is in a vector, which may be a plasmid or a virus, such as adenovirus or retrovirus.
  • the transfect ion may occur ex vivo or in vivo by direct injection of the isolated nucleic acid molecule.
  • direct administration of the SIP30 antagonist is possible.
  • the inventors discover a process for determining suitable SIP30 antagonists for their ability to interact with SIP30-like molecule protein, including SNAREs comprising: a) providing a SIP30-like molecule polypeptide comprising the contiguous amino acid sequence of any from the group of SEQ. ID. Bl through SEQ. ID. B130; and b) testing the ability of said substance to interact with a SIP30-like molecule.
  • the step of testing the ability of said substance to interact with a SIP30-like molecule involves determining the interaction affinity of said substance to a SIP30-like molecule.
  • the inventors disclose a process for determining a substance's ability to interact with a SIP30-like molecule comprising: a) providing a recombinant SIP30-like molecule polypeptide encoded by a nucleic acid sequence comprising at least 35 contiguous nucleotides of any from the group of SEQ. ID. Al through SEQ. ID. A130; b) contacting said substance with said recombinant SIP30-like molecule polypeptide; and c) detecting the ability of said substance to bind to said recombinant SIP30-like molecule polypeptide.
  • the nucleic acid sequence comprises at least 45 contiguous nucleotides of any from the group of SEQ.
  • the nucleic acid sequence comprises at least 50, 75, or 100 contiguous nucleotides of any from the group of SEQ. ID. Al through SEQ. ID. A130.
  • Another aspect of the invention is directed to processes for detecting the ability of suitable SIP30 antagonists to interact with a SNAREs and SIP30 molecule polypeptide by measuring a) the ability of said recombinant SIP30-like molecule polypeptide to interact with said substance; b) the ability of said substance to activate ion channels in a cell membrane; or c) modulation of channels in the cell membrane of part b) , specifically, wherein the recombinant SIP30-like molecule polypeptide is chimeric.
  • Another aspect of the present invention is directed processs for determining a substance's ability to bind to SIP30-like molecule comprising: a) expressing in cells a recombinant SIP30 polypeptide encoded by a nucleic acid sequence comprising at least 25 contiguous bases of any from the group of SEQ ID Al through SEQ ID A130; b) contacting said substance with said recombinant SIP30-like molecule polypeptide; and c) detecting the ability of said substance to interact with said recombinant SIP30-like molecule polypeptide.
  • the nucleic acid sequence can comprise at least 40, 45, 50, 75 or 100 contiguous nucleotides of any from the group of SEQ ID Al through SEQ ID A130.
  • a preferred embodiment the present invention provides a an antisense oligodeoxynucleotide complementary to nucleotide 11031 of the coding region of the rat SIP30.
  • Another aspect of the present invention is directed to siRNA molecule corresponding to at least a portion of a SIP30 nucleic acid sequence, mRNA encoding SNAREs, including SNAP25 capable of inhibiting expression of said proteins or cytokines in a cell responsible for neuropathic phenomenon.
  • siRNAs of the present invention are typically short
  • the siRNA molecules comprise a double-stranded structure comprising a sense strand and an antisense strand, wherein the antisense strand comprises a nucleotide sequence that is complementary to at least a portion of a peptide, protein or cytokine nucleic acid sequence and the sense strand comprises a nucleotide sequence that is complementary to at least a portion of the nucleotide sequence of said antisense region, and wherein the sense strand and the antisense strand each comprise about 19-29 nucleotides.
  • the target segment of the target mRNA preferably should begin with AA (most preferred) , TA, GA, or CA; the GC ratio of the siRNA molecule preferably should be 45-55%; the siRNA molecule preferably should not contain three of the same nucleotides in a row; the siRNA molecule preferably should not contain seven mixed G/Cs in a row; the siRNA molecule preferably should comprise two nucleotide overhangs (preferably TT) at each 3' terminus; the target segment preferably should be in the ORF (open reading frame) region of the target mRNA and preferably should be at least 75 bp after the initiation ATG and at least 75 bp before the stop codon; and the target segment preferably should not contain more than 16-17
  • another aspect of the present invention provides a method for inhibiting expression of a SIP30 pain inducing protein or cytokine, or other intermediaries in a cell comprising introducing into a cell at least one antisence or siRNA molecule that corresponds to at least a portion of a pain inducing nucleic acid sequence.
  • the cell into which the such molecules should be introduced is preferably a central nervous system regions that at risk of SIP30 upregulation, more preferably a spinal cord or locally at areas that the pain inducing protein or cytokines manifest their presence.
  • the regions is from a subject suffering from an injury, preferably a human patient.
  • the compounds and molecules produced herein can be introduced into cells in vitro or ex vivo using techniques well-known in the art, including electroporation, calcium phosphate co-precipitation, microinjection, lipofection, polyfection, and conjugation to cell penetrating peptides (CPPs) .
  • CPPs cell penetrating peptides
  • such molecules targeted to pain inducing cytokines can be introduced into cells in vivo by endogenous production from an expression vector (s) encoding the sense and antisense sequences.
  • Genetic control elements include a transcriptional promoter, and may also include transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription.
  • Suitable eukaryotic promoters include constitutive RNA polymerase II promoters (e.g., cytomegalovirus (CMV) promoter, the SV40 early promoter region, the promoter contained in the 3 ' long terminal repeat of Rous sarcoma virus (RSV) , the herpes thymidine kinase (TK) promoter, and the chicken beta-actin promoter) , tissue-specific RNA polymerase II promoters, and RNA polymerase III promoters (e.g., U6, HI, 7SK and 7SL) .
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • TK herpes thymidine kinase
  • the sense and antisense strands may be encoded by different expression vectors (i.e., co- transfected) .
  • the sense and antisense strands of siRNA molecules are encoded by the same expression vector.
  • the sense and antisense strands can be expressed separately from a single expression vector, using either convergent or divergent transcription.
  • the sense and antisense strands can be expressed together from a single expression vector in the form of a single hairpin RNA molecule, either as a short hairpin RNA (shRNA) molecule (e.g., Arts et al . , Genome Res. 13:2325 (2003)) or a long hairpin RNA molecule (e.g., Paddison et al . , Proc. Natl. Acad. Sci. USA 99:1443 (2002)).
  • shRNA short hairpin RNA
  • a long hairpin RNA molecule e.g., Paddison et al . , Proc. Natl. Acad. Sci. USA 99:1443 (2002).
  • cytokine polypeptide (or fragments thereof) can be detected and quantified using various well-known immunological assays, such as, e.g., enzyme-linked immunosorbent assay (ELISA) , radio-immunoassay (RIA) , immunoprecipitation, immunofluorescence, and Western blotting.
  • immunological assays such as, e.g., enzyme-linked immunosorbent assay (ELISA) , radio-immunoassay (RIA) , immunoprecipitation, immunofluorescence, and Western blotting.
  • Anti-pro-inflammatory cytokine anti-bodies for use in immunological assays are commercially available from, e.g., EMD Biosciences (San Diego, CA) , Upstate (Charlottes-ville, VA) , Abeam (Cambridge, MA) , Affinity Bioreagents (Golden, CO) and Novus Biologicals (Littleton, CO) , or may be produced by methods well-known to those skilled in the art.
  • the invention provides a screening method comprising: providing a sample containing at least a portion of SIP30 and/or at least a portion of a SIP30 protein; adding a test compound to at least a first portion of the sample; and comparing at least one parameter from at least the first portion of the sample with the at least one parameter from at least a second portion of the sample, wherein at least the second portion of the sample does not include the test compound.
  • a magnitude of a difference between the at least one parameter from at least the first portion of the sample and the at least one parameter from at least the second portion of the sample is an indication of, inter alia, the test compound's ability to alter SIP30 expression, activity or up-regulation .
  • the invention provides a kit comprising at least a portion of SIP30 and/or at least the portion of SIP30 protein, and at least a quantity of a control compounds.
  • the invention provides a method for use in assessing the risk of developing post surgical neuropathic pain in a subject comprising detecting a test amount of a SIP30 gene product in a sample from the subject; and comparing the test amount with a normal amount of SIP30 gene product in a control sample, whereby a finding that the test amount is outside of the normal range for the SIP30 gene product provides a positive indication in the diagnosis of a risk to post surgical neuropathic pain.
  • the use of antisense or siRNA molecules alone or in combination with other anticonvulsants, anti-inflammatory agents, anesthetics, to inhibit cellular expression of neuropathic pain inducing cytokine finds utilities as methods for the treatment inflammation in subjects.
  • another aspect of the present invention provides a method for treating a patient suffering from neuropathic pain comprising introducing into said patient at least an effective amount of SIP30 antagonist molecule that corresponds to at least a portion of a SIP 30 nucleic acid sequence.
  • a method for treatment of inflammation can be performed using systems that provide for the delivery of such molecules targeted to the tissue of interest.
  • another aspect of the present invention provides a means for introducing the effective amount of such formulations into the tissue of the patient.
  • the patient is human.
  • a preferred delivery system includes a depot implant.
  • a depot implant of the present invention comprises a physical structure to facilitate implantation and retention in a desired location of a subject.
  • Drug depot implant may contain microspheres, the microspheres may further contain a isolated nucleic acids, a siRNA molecule that provides a concentration gradient for targeted delivery of the agent to the subject.
  • the microspheres are injected into the tissue of interest.
  • the practitioner may formulate the at least one SIP30 antagonist as a combination of a gel and microspheres loaded with the at least one anticonvulsant, antidepressant, anti-inflammatory, opioid, or anesthetic wherein the combination of gel and microspheres are placed in the region of interest.
  • the administration is localized and sustained.
  • Example 1 Antagonizing SIP30 to suppress development and maintenance of peripheral nerve in ury-induced neuropathic pain
  • Intrathecal implantation An intrathecal catheter (PE-10 tube) was inserted through the gap between the L4 and L5 vertebrae and extended to the subarachniod space of the lumbar enlargement (L4 and L5 segments) under sodium pentobarbital (40 mg/kg, i.p.) anesthesia.
  • the catheter was filled with sterile normal saline (approximately 4 ul) , and the outer end was plugged.
  • the cannulated rats were allowed to recover for 4 days and were housed individually. Rats that showed any neurological deficits resulting from the surgical procedure were excluded from the experiments.
  • CCI Chronic constriction injury
  • Rats were deeply anesthetized with pentobarbital sodium (45 mg/kg i.p.) and the right sciatic nerve exposed at the mid-thigh level by blunt dissection of the biceps femoris.
  • pentobarbital sodium 45 mg/kg i.p.
  • the right sciatic nerve exposed at the mid-thigh level by blunt dissection of the biceps femoris.
  • 4-0 chromic gut
  • ligatures were tied loosely around the nerve ⁇ 1 mm apart, proximal to its trifurcation, as described by Bennett and Xie (1988) .
  • the sciatic nerve was isolated but not ligated. After CCI or sham surgery, the overlying muscles and skin were closed respectively in layers with 4-0 silk sutures and dusted with antibiotic power.
  • Antisense oligodeoxynucleotide (ODN) preparation and delivery An antisense sequence (AS, 5'- TTT CTC CGC GTC CGC CAT GGT -3') SEQ. ID No. B131, complementary to nucleotides 11-31 of the coding region of the rat SNAP25 interacting protein 30 (SIP30, GenBank accession number BC063144), and a corresponding mismatch sequence SEQ. ID. No. B132 (MM, 5' -TTT CCT CGC GTC CCG CAT GGT- 3') were synthesized and purified by Integrated DNA Technologies, Inc., Coralville, IA, USA).
  • ODNs were screened against the GenBank Database using the BLAST algorithm to exclude non- specificity of the antisense ODNs and to show that missense ODNs did not match any registered nucleotide sequences.
  • the ODNs were reconstituted in 0.9 % normal saline (NS) before administration.
  • the rats were injected intrathecally with NS (10 ul), AS (50 ug/10 ul), and MM (50 ug/10 ul), respectively, followed by 5 ul NS for flushing, every 24 hrs for 4 days [delivering protocol I: from day o (6 hrs before CCI operation) to day 3 post-CCI operation; delivering protocol II: from day 4 to day 7 post-CCI operation] .
  • RNA extraction for real-time PCR analysis Rats were sacrificed on the 3rd or 7th day after CCI or sham operation. In order to assess the development of neuropathic pain and effects of antisense, rats were tested for both mechanical allodynia and thermal hyperalgesia as described in 2.6 before sacrificed.
  • the spinal cord from naive, sham, or CCI rats treated with AS, MM, or NS was dissected and split into two halves, one ipsilateral and one contralateral to the CCI side.
  • the DRG (L4-L6) from the same treatment animals were also dissected. A single sample of the ipsilateral or contra-lateral DRG was collected from two rats.
  • Real-time PCR Real-time PCR analysis was performed on a Cepheid Smart Cycler. The threshold cycles (Ct) were calculated using the Smart Cycler Data Analysis Software 2.0 (Cepheid) .
  • the PCR reaction was performed in a final volume of 20 ⁇ using LightCycler DNA Master SYBR Green I kit containing: 12.6 ⁇ of PCR grade water, 2.4 ⁇ of MgC12 (25 mM) , 2 ⁇ of 10x LightCycler DNA Master SYBR Green I [Taq DNA polymerase, reaction buffer dNTP mix (with dUTP instead of dTTP) , SYBR Green I dye, and 10 mM MgC12], 2 ⁇ of cDNA template, 0.5 ⁇ of forward and reverse primer (100 ng/ ⁇ ) respectively .
  • the amplification protocol included 150 sec at 95 °C to activate the Taq DNA polymerase, then 40 cycles of 10 sec denaturation at 95 °C, 15 sec annealing at 58 °C, and 20 sec extension at 72 °C. In all cases, a linear relationship between the sample cDNA quantity and the Ct was observed first. Each sample was run in triplicates and Ct values were averaged from each reaction.
  • the hind paw withdrawal threshold was determined using a calibrated series of von Frey hairs (Stoelting, IL, USA) ranging from 0.6 to 18 g. Animals were placed individually into wire mesh-bottom cages, and allowed to acclimatize for -30 min. The series of von Frey hairs was applied to the central region of the plantar surface of the left hind paw in ascending order of force (0.6, 0.9, 1.3, 2.2, 4.8, 6, 7.2, 9, 13, and 18 g) with a single trial of up to 6 sec. The hair was applied only when the rat was stationary and standing on all four paws. A withdrawal response was considered valid only if the hind paw was completely removed from the mesh-bottom.
  • the paw was retested, starting with the next descending von Frey hair until no response occurred.
  • a trial consisted of application of a von Frey hair to the hindpaw five times at 15 s intervals to each hindpaw.
  • the hindpaw withdrawal threshold was defined as the lowest force that caused at least three withdrawals out of the five consecutive applications. Once the threshold was determined for the left hind paw, the same testing procedure was repeated on the right hind paw after 5 min.
  • This time was defined as the hind paw withdrawal latency.
  • the heat was maintained at a constant intensity, which produced a stable withdrawal latency of approximately 8-10 s in the absence of CCI .
  • a 20 s cut-off was used to prevent tissue damage in the absence of a response. Rats were tested individually in groups of four, such that the stimulation was delivered once to the left hind paw (control) of each rat and then to the right hind paw (CCI) with a 5 min interval. This process was then repeated two more times, and three trials were averaged for each limb.
  • CCI chronic constriction injury
  • RNA from the lumbar enlargement portion of the control and CCI animals was used to perform microarray analysis using a custom-constructed cDNA library microarray as previously described (Hou et al . , 2004; Jin et al . , 2005; Wang et al . , 2005) .
  • One of the cDNA clones that were identified in this process is SIP30, a molecule that has been previously reported that interacts with SNAP25 (Lee et al . , 2002), which has been shown to be present in several areas of the rat brain (Lee et al . , 2002), and its molecular function was hitherto unknown.
  • SIP30 orthologues are present in several species, including chimpanzee, monkey, and several non-primate mammals.
  • the sequence is highly homologous. Extensive search of GenBank and other public sequence databases indicated that SIP30 sequence is present in primates and other mammals.
  • Small-diameter DRG neurons are primarily nociceptors. They can be divided neurochemically into two populations: isolectinB4 ( IB4 ) -positive nonpeptidergic neurons, and IB4- negative peptidergic neurons.
  • the IB4 negative neurons express TrkA receptors for nerve growth factor (NGF) , depend on NGF for survival, and contain neuropeptides including calcitonin gene-related peptide and substance P.
  • the IB4 positive neuronal population expresses receptors for glial- derived neurotrophic factor (GDNF) , neurturin, or artemin.
  • GDNF glial- derived neurotrophic factor
  • Isolectin B4-positive and -negative nociceptors are functionally distinct (Stucky and Lewin, 1999) . It has been hypothesized that IB4-negative neurons specifically contribute to inflammatory pain and that IB4-positive neurons contribute to neuropathic pain (Mantyh and Hunt, 1998; Snider and McMahon, 1998; Breese et al . , 2005).
  • SIP30 expression was examined together with IB4 and CGRP in normal adult rats DRG (L4-L5) .
  • SIP30 is expressed across small to medium diameter DRG neurons ( Figure 3) .
  • SIP30 displayed an overlapping pattern with IB4 and CGRP in some neurons but mainly co-expressed with CGRP ( Figure 3), suggesting a peptidergic nature.
  • SIP30 immunoreactivity was examined in the lumbar spinal cord, where it was found to be expressed in all lumbar levels and throughout all laminae. SIP30 immunoreactivity was particularly concentrated in the superficial dorsal horn lamina I and II ( Figure 3), and showed a strong co-expression pattern with nociception- related neuropeptides CGRP and substance P ( Figure 3) .
  • SIP30 immunostaining was present in cell bodies in laminae IV-VII as well as in motor neurons in the ventral horn .
  • Upregulation of SIP30 in the spinal cord following CCI was measured by quantitative real ⁇ time PCR, in both spinal cord and DRG of either CCI or sham- operated rats. The expression levels of two other genes were used as reference points to normalize the amount of total RNA amount in each sample. The expression of ⁇ -actin and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) among the intact and CCI rats' spinal cord and DRG using quantitative real-time PCR analysis were first examined. The ⁇ -actin and GAPDH genes expression of the CCI rats was normalized to those of intact rats.
  • GAPDH glyceraldehydes-3-phosphate dehydrogenase
  • SIP30 protein was also demonstrated by western blotting analysis of the spinal cord from CCI rats. Increased signal for SIP30 was observed in ipsilateral lumbar spinal cord compared with the contralateral side of CCI rats and both side of sham-CCI animals ( Figure 4) . Similar to the expression of SIP30 mRNA in the DRG from CCI and Sham rats, no significant changes in western blotting signal for SIP30 were found on both sides of the DRG following CCI ( Figure 4) .
  • SIP30 localization in CCI spinal cord was examined, showing colocalization with NeuN, a neuronal marker, in the spinal dorsal horn, 3 days after CCI ( Figure 4) .
  • SIP30 did not show overlap staining with either GFAP or OX-42 (Fig. 4) .
  • SIP30 is involved in the development and maintenance of neuropathic pain.
  • - Intrathecal administration of SIP30 AS (50 mg/10 ul, once a day for 4 days) starting on day 0 (6 hrs before CCI), produced a partial inhibition in the development of mechanical allodynia and thermal hyperalgesia by day 4 of SIP30 AS administration ( Figure 5) .
  • Two-way ANOVA showed a statistically significant difference between the antisense group and NS group (p ⁇ 0.05) .
  • Two days after cessation of AS administration post-CCI day 5
  • the AS group showed mechanical allodynia similar to the NS group ( Figure 5) .
  • Anti-SIP30 oligonucleotide knock-down inhibits both SIP30 and SNAP25 expression in the spinal cord- Considering the CCI procedure failed to affect the expression of SIP30 in the DRG at either mRNA or protein level, the following observations were focused only on the spinal cord.
  • SIP30 AS ODN indeed results in knockdown of SIP30 by disrupting the translation of SIP30 mRNA
  • SIP30 AS ODN was assessed.
  • SIP30 AS 50 ug/10 ul
  • MM ODN 50 ug/10 ul
  • SNAP25 mRNA exhibited significant up-regulation in the ipsilateral spinal cord on both day 3 and 7 post-CCI .
  • PSD95 mRNA in the spinal cord had no detectable change until day 7 post-CCI.
  • SIP30 AS nor MM ODN blocked the upregulation of PSD95 mRNA in the ipsilateral spinal cord on day 7 after CCI (Fig. 6) .
  • Example 2- Role of SIP30 in intensifying pain sensitivity
  • mice were subjected to intrathecal administration of SIP30 and control reagents, in the form of plasmid DNA. Either plasmid containing SIP30 sequence or empty plasmid were used, together with a lipid transfection reagent.
  • the mice were tested for paw withdraw threshold via Von Frey filament over the next 24 days.
  • Figure 7 depicts the outcome of the experiment. Mice in both SIP30 groups A and B showed significant change in Paw Withdraw Threshold from control group of mice at testing days 4, 6, and 8 (*, p ⁇ 0.05 significant difference compared to control) . Measurements included both legs from each mouse. By day 24, paw withdraw threshold had returned to normal levels. Those of ordinary skill in the art in possession of the present invention would appreciate the direct role of SIP30 in aggravating the neuropathic pain. The present findings provide ample guidance as to the duration and degree of SIP30 antagonism that is required to provide a therapeutic regimen.

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Abstract

La présente invention concerne des composés, des compositions, des procédés, des systèmes et des nécessaires pour le traitement de la douleur névropathique régulée par SIP30. La présente invention concerne des antagonistes de SIP30 pour le traitement de la douleur névropathique.
PCT/US2010/047657 2009-09-02 2010-09-02 Compositions et procédés de traitement de la douleur névropathique WO2011028890A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005014849A2 (fr) 2003-07-03 2005-02-17 Euro-Celtique, S.A. Genes associes a des reponses a des douleurs neuropathiques

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Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. BC063144
ARTS ET AL., GENOME RES., vol. 13, 2003, pages 2325
AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 1994
BASS, NATURE, vol. 411, 2001, pages 428
ELBASHIR ET AL., EMBO J., vol. 20, 2001, pages 6877
PADDISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 99, 2002, pages 1443
PENG GET: "SIP30 Is Regulated by ERK in Peripheral Nerve Injury induced Neuropathic Pain", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 284, no. 44, October 2009 (2009-10-01), pages 30138 - 30147, XP002689803, DOI: 10.1074/jbc.M109.036756
See also references of EP2473635A4
SOHAIL, GENE SILENCING BY RNA INTERFERENCE: TECHNOLOGY AND APPLICATION, 2004
ZHANG ET AL.: "Role of SIP30 in the development and maintenance of peripheral nerve injury-induced neuropathic pain", PAIN, vol. 146, 13 July 2009 (2009-07-13), pages 130 - 140, XP026677409, DOI: 10.1016/j.pain.2009.07.011

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CN107090027A (zh) 2017-08-25
CN102666877A (zh) 2012-09-12
CN102666877B (zh) 2018-06-12
US20120245216A1 (en) 2012-09-27
CA2772931A1 (fr) 2011-03-10
CN107121554A (zh) 2017-09-01
US9068984B2 (en) 2015-06-30
EP2473635A4 (fr) 2013-03-13
EP2473635A2 (fr) 2012-07-11
IL218448A0 (en) 2012-04-30
WO2011028890A3 (fr) 2011-05-05

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